PROJECT SUMMARY Muscle invasive urothelial bladder cancer is a clinically aggressive disease with poor outcomes despite modern therapies. Recently, antibodies targeting the PD-1/PD-L1 immune checkpoint pathway have shown efficacy and were approved for use for locally advanced or metastatic bladder cancer. In some patients, impressive and durable clinical responses are observed, yet the rate of response remains modest at 20%. Mechanisms of pri- mary immune resistance in bladder cancer are not fully understood. The presence of a T cell-inflamed tumor phenotype has been associated with improved survival and response to immunotherapies. T cell-inflamed tu- mors can be identified by the presence of intratumoral CD8+ T cells or expression of a T cell gene signature. Emerging data have indicated that certain oncogenic pathways, such as FGFR3, are activated exclusively in non-T cell-inflamed bladder cancers. The underlying hypothesis of this proposal is that FGFR3 activation pre- vents spontaneous T cell priming in bladder cancer and may be therapeutically targeted to restore sensitivity to immune checkpoint blockade. A mechanistic investigation into the immunomodulatory effects of FGFR3 will be pursued through (a) a new genetically-engineered mouse model system with tamoxifen-inducible KrasG12D/p53fl/fl carcinomas that also express an activating FGFR3K644E mutation, (b) a syngeneic transplantable mouse model system using the MB49 bladder cancer cell line with model antigen expression, (c) in vitro studies examining differential gene expression in CRISPR-Cac9 derived FGFR3 knockout compared with FGFR active tumor cells, and (d) by an analysis of the immune microenvironment and FGFR3 expression in human bladder specimens using RNA in situ hybridization and multichannel immunofluorescence. The predictive capacity of these param- eters will be assessed in relation to outcomes for patients treated with anti-PD-1/L1 immunotherapy. Dr. Sweis is a physician-scientist from the University of Chicago and is the principle investigator on this study. He has devised a career development plan to obtain training in advanced tumor immunology and laboratory mouse modeling in order to facilitate his development as an independent investigator. He has developed a mentoring committee with broad expertise that is led by Dr. Thomas Gajewski, an internationally renowned expert in cancer immunology. Dr. Sweis has dedicated his efforts to the investigation of immunotherapy resistance mechanisms to improve outcomes for patients.